direct analysis of multicomponent adjuvants by hplc with ......of analysis including quantification...

Direct Analysis of Multicomponent Adjuvants by HPLC with Charged Aerosol DetectionDavid Thomas, Ian Acworth, Bruce Bailey, Marc Plante Thermo Fisher Scientific, Chelmsford, MA, USA

2 Direct Analysis of Multicomponent Adjuvants by HPLC with Charged Aerosol Detection

Direct Analysis of Multicomponent Adjuvants by HPLC with Charged Aerosol DetectionDavid Thomas, Ian Acworth, Bruce Bailey, Marc PlanteTh Fi h S i tifi Ch l f d MAThermo Fisher Scientific, Chelmsford, MA

FIGURE 4. Chromatograms of AddaVax and emulsifiers by HPLC-charged aerosol detection.

Table 2. Summary of immunologic adjuvant components investigated in thisstudy. (+) annotation indicates applicable detector.Methods

Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system with a Diode Array Detector DAD 3000 RS and a Corona™ ultra RS™ Charged Aerosol Detector:

Component UV(220 nm)

Charged Aerosol120

140

160

squalene

800

900

1000

ResultsAbISCO-100

AbISCO-100 is a suspension of purified saponins from Quillaja saponaria, cholesterolfrom sheep wool and egg phosphatidyl choline in phosphate buffered saline1. As seen in Figure 2, all components elute within 12 min from the Hypersil GOLD PFP column

ith d l ti All t d l d d ti d t i l di

OverviewPurpose: To develop fast and sensitive HPLC methods suitable to measure the purity of immunological adjuvant formulations.

Methods: Adjuvant mixtures and individual components were analyzed by ultra-high performance liquid chromatography (UHPLC) employing octylsilyl (C8) and

fl h l (PFP) t ti h ith b 2 i ili Corona ultra RS Charged Aerosol Detector:Nebulizer Temperature: 25 °CPower function: 1.00Data collection rate: 20 Hz

Reagents: HPLC- or LCMS-grade or better

Data Analysis

CDS: Thermo Scientific Dionex Chromeleon™ ChromatographyData System 7 1 SR1

Saponins + +

Cholesterol + +

Diphosphatidyl choline (DPPC) +

Mixed phosphatidyl cholines +

Lyso-phosphatidyl choline +20

40

60

80

100

200

300

400

500

600

700

20.0pA mAU

200

with good resolution. All components and several degradation products including cholesterol oxidation products (COP) and lyso-PC are detected by the charged aerosoldetector, whereas some, such as DPPC, show poor response by UV detection.

FIGURE 2. Charged aerosol response is significantly better than UV response for several components of AbISCO-100 separated by HPLC.

perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or 2.6 micron solid core silica particles. Analytes were detected by both charged aerosol detection and photodiode array detection.

Results: Charged aerosol detection is shown to be suitable for measuring components including triterpenoid saponins, cholesterol, phospholipids, and surfactants with sensitivity often superior to that obtained by UV absorbance detection.

Introduction

AddaVax

polysorbate 80

Data System 7.1 SR1

AbISCO-100Column: Thermo Scientific Hypersil GOLD™ PFP 1.9 µm,

2.1 × 100 mmColumn Temp: 45 °CFlow Rate: 0.47 mL/minInjection Vol.: 10 µLMobile Phase A: 0 1% formic acid in water

Sorbitan trioleate (Span 85) +

Polysorbate 80 (Tween 80) +

Squalene + +

α-Tocopherol + +

sMPLA + +

Squalene-based mixture

Squalene is mixed with other components in several other adjuvants. The chromatogram shown in Fig 5 illustrates the results obtained with a mixture of

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180IntroductionA vaccine adjuvant is any substance that helps promote the effectiveness of a vaccine by reducing the amount or frequency of the required dose, by prolonging the duration of immunological memory, or by modulating the involvement of humoral or cellular responses. This functional definition of adjuvants encompasses a very diverse group of substances whose chemical structures and mechanisms of action vary widely. Adjuvants for human or animal vaccines are typically subjected to rigorous standards

Span-85

cholesterol

Conclusion The HPLC method developed to analyze AbISCO is precise, with retention time

precision better than 0.1% RSD and peak area precision between 0.4 and 1.3% RSD for the major components.

Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in 10:90 acetonitrile:reagent alcoholGradient: Time, %B: -5, 35; 0, 35; 7, 80; 12, 80.Sample Prep.:ABISCO-100™ (ISCONOVA, Uppsala Sweden) was diluted 5-fold with Milli-Q® waterand transferred to a glass HPLC autosampler vial.

AddaVax

FIGURE 5. HPLC-charged aerosol detection chromatogram of a mixture of α-tocopherol, squalene and PS 80.

squalene, DL-α-tocopherol and polysorbate 80 (PS 80).

8.0

9.0

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alpha-tocopherol

pA pA

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100

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of analysis including quantification of strength, purity, stability, and degradation behavior, even though they are not currently regulated in the same manner as active pharmaceutical ingredients in the United States. Complicating such analysis, many adjuvants under investigation contain components that are not readily analyzed by traditional HPLC with UV detection. These include various mixtures of lipids, fatty acids, and glycosides that lack suitable UV chromophores. In this work, the lack of a detectable chromophore in several adjuvant compounds and degradation products was overcome by using HPLC with charged aerosol detection, a detector that can measure

cholesterol

phosphatidylcholinesaponins

lyso‐PC COP

Charged aerosol detection enables sensitive measurement of adjuvantcomponents not amenable to detection by UV absorbance. Detection limits forsaponins, cholesterol, and DPPC were in the low µg/mL (ng on-column) range.

By responding uniformly to structurally diverse compounds, charged aerosol

Column: Thermo Scientific Accucore™ C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °CFlow Rate: 0.80 mL/minInjection Vol.: 10 µLMobile Phase A: 1 mM ammonium acetate in waterMobile Phase B: 2-propanol, Fisher Scientific™ Optima™2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.: 2.0

3.0

4.0

5.0

6.0

7.0 squalene

20

30

40

50

60

70Performance

Calibration curves for the three major components of AbISCO (analyzed in duplicate)are presented in Figure 3. The data were fit to a quadratic equation, yielding coefficients of determination, R2, greater than 0.999 for all three analytes.

Table 1 presents a summary of the method’s performance, including precision ofretention time and peak area, the coefficient of determination, and the limits ofdetection for the three major components of AbISCO.

y g g ,any non-volatile compound.

Adjuvant mixtures and individual components were analyzed by UHPLC employing octylsilyl (C8) and perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or 2.6 micron solid core silica particles. Analytes were detected by both charged aerosol detection and diode array detection.

The charged aerosol detector is a sensitive, mass-based detector, especially well-suited for the determination of any nonvolatile analyte independent of chemical

mixturel y p g y y p , g

detection is able to measure intact adjuvant species along with degradation products and potential impurities, yielding good estimates of relative concentration, even in the absence of pure primary standards.

References1 Pi d M D C h K P Gi h T M Hi i D E Fl ht J B Hi h

p pAddaVax™ (InvivoGen, San Diego, CA) was diluted 10-fold with Milli-Q water and transferred to a glass HPLC autosampler vial.

Squalene-tocopherol-PS80 mixtureColumn: Thermo Scientific Accucore PFP 2.6 µm, 2.1× 100 mm Column Temp: 45 °CFlow Rate: 0.50 mL/minInjection Vol : 1 or 3 µL

sMPLA

Synthetic monophosphoryl lipid A, an analog of bacterial lipopolysaccharide (LPS), has

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min-5

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10

j p

Cholesterol External CAD_1pA*min

3.50

4.00

4.50

5.00

5.50 DPPC External CAD_1pA*min

5.00

6.00

7.00saponin 3 External CAD_1pA*min

4.00

4.50

5.00

5.50

6.00

Figure 3. Calibration data for analysis of AbISCO by HPLC-charged aerosoldetection.

suited for the determination of any nonvolatile analyte independent of chemical characteristics. As shown in Figure 1, the detector nebulizes the mobile phase to create aerosol droplets. The mobile phase evaporates in the drying tube, leaving analyte particles, which become charged in the mixing chamber. The charge is then measured by a highly sensitive electrometer, providing reproducible, nanogram-level sensitivity. This technology has greater sensitivity and precision than evaporative light scattering detection and refractive index detection and it is simpler to operate than a mass spectrometer.

squaleneα‐tocopherol

1. Picard, M.D.; Cohane, K.P.; Gierahn, T.M.; Higgins, D.E.; Flechtner, J.B. High-throughput proteomic screening identifies Chlamydia trachomatis antigens thatare capable of eliciting T cell and antibody responses that provide protection against vaginal challenge. Vaccine. 2012, 30(29):4387-93.

2. Mbow M.L.; De Gregorio, E.; Valiante, N.M.; Rappuoli, R. New adjuvants forhuman vaccines. Curr Opin Immunol. 2010, 2(3):411-6.

3. Raetz, C.R.; Garrett, T.A.; Reynolds, C.M. et al. Kdo2-Lipid A of Escherichia coli,

FIGURE 1. Charged Aerosol Detector and Principle of Operation

HPLC eluent

Nebulization and

Drying tube

Signal out

Injection Vol.: 1 or 3 µLMobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.08% formic acid in 2-propanol, Optima 2 LC/MSGradient: Time, %B: -5, 30; 0, 30; 13, 95; 18, 95 Sample Prep.:Dissolve 24.3 mg DL-α-tocopherol (Acros Organics, NJ) in 24.3 mL 2-propanol.Dissolve 48.7 mg squalene in 9.64 mL 2-propanol; dilute 5-fold with 2-propanol.Combine 214 µL squalene solution, 237 µL DL-α-tocopherol solution, 97 µLpolysorbate 80 and 452 µL of 2 propanol

FIGURE 6. Purity analysis of sMPLA by HPLC-UV-charged aerosol detection.

y p p y p , g p p y ( ),low toxicity while specifically activating TLR4 but not TLR2. sMPLA contains six acylgroups. Purity analysis is used to quantify contaminants, degradation products, and variants differing in acyl chain number, length, and phosphorylation3. Figure 6 compares chromatograms obtained from analysis of sMPLA by inverse gradient HPLC with detection by UV absorbance at 220 nm or charged aerosol detection.

Table 1. Method performance for analysis of AbISCO by HPLC-charged aerosol

ug/mL

0 50 100 150 200 250 300Amount

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0.50

1.00

1.50

2.00

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3.00

Area

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3.50

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a defined endotoxin that activates macrophages via TLR-4. J. Lipid Res. 2006, 47:1097-1111.

AcknowledgementsWe would like to thank Isconova, Uppsala, Sweden for making available the AbISCO

Nebulization andImpactor

polysorbate 80 and 452 µL of 2-propanol.

Synthetic MPLAColumn: Thermo Scientific Accucore C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °CFlow Rate: 0.80 mL/minInjection Vol.: 10 µLMobile Phase A: 1 mM ammonium acetate in waterM bil Ph B 2 l O ti 2 LC/MS

p y y gdetection.

Component Amount(µg/mL)

Ret. Time1

(%RSD)Peak Area1

(%RSD)LOD2

µg/mLR2*

Saponins 78.2 0.06 0.36 13.0 0.9998

Cholesterol 56.8 0.07 0.39 6.4 0.9999

DPPC 60 2 0 07 1 27 4 4 1 000025.0

30.0

35.0

40.0

45.0

50.0pA mAU

25.0

30.0

35.0

40.0

45.0

50.0

sMPLA We would like to thank Isconova, Uppsala, Sweden for making available the AbISCOimmunological reagent.

AddaVax is a trademark of InvivoGen, San Diego, CA. AbISCO-100 is a registered trademark of ISCONOVA,Uppsala, Sweden. Span 85 and Tween 80 are trademarks of Croda International Plc. Milli-Q is a registered trademark of EMD Millipore Corporation. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

Thi i f ti i t i t d d t f th d t i th t i ht i f i th

AddaVax

AddaVax, shown in Figure 4, is prepared by emulsification of sorbitan trioleate

Liquid waste

Dried particlesCorona wire

Mixing chamber

Ion trap

Collector / Electrometer

Mobile Phase B: 2-propanol, Optima 2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.:Synthetic MPLA (Avanti Polar Lipids, GA) was dissolved in chloroform:methanol:water(CMW) 80:20:4 at a concentration of about 1 mg/mL and transferred to a glass HPLCautosampler vial.

DPPC 60.2 0.07 1.27 4.4 1.00001 for n = 10 replicates

2 Hubaux-Vos method

* 7 levels, in duplicate, quadratic fit with no offset

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10.0

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Inverse gradientStandard gradient

sMPLA

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70333_E 10/12S

AddaVax, shown in Figure 4, is prepared by emulsification of sorbitan trioleate(SPAN™ 85) in squalene oil (5% v/v) and polysorbate 80 (0.5% w/v) in sodium citrate buffer (10 mM pH 6.5)2.

Time [min]

3Thermo Scientific Poster Note • PN70333_e 11/12S







Charged Aerosol120

140

160

squalene

800

900

1000

ResultsAbISCO-100



OverviewPurpose: To develop fast and sensitive HPLC methods suitable to measure the purityof immunological adjuvant formulations.

Methods: Adjuvant mixtures and individual components were analyzed by ultra-highperformance liquid chromatography (UHPLC) employing octylsilyl (C8) and



Data Analysis

CDS: Thermo Scientific Dionex Chromeleon™ Chromatography Data System 7 1 SR1

Saponins + +

Cholesterol + +




40

60

80

100

200

300

400

500

600

700

20.0pA mAU

200



perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or2.6 micron solid core silica particles. Analytes were detected by both charged aerosoldetection and photodiode array detection.

Results: Charged aerosol detection is shown to be suitable for measuring componentsincluding triterpenoid saponins, cholesterol, phospholipids, and surfactants with sensitivity often superior to that obtained by UV absorbance detection.

Introduction

AddaVax

polysorbate 80

Data System 7.1 SR1


2.1 × 100 mmColumn Temp: 45 °C Flow Rate: 0.47 mL/minInjection Vol.: 10 µL Mobile Phase A: 0 1% formic acid in water



Squalene + +

α-Tocopherol + +

sMPLA + +



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180IntroductionA vaccine adjuvant is any substance that helps promote the effectiveness of a vaccine by reducing the amount or frequency of the required dose, by prolonging the duration of immunological memory, or by modulating the involvement of humoral or cellularresponses. This functional definition of adjuvants encompasses a very diverse group of substances whose chemical structures and mechanisms of action vary widely.Adjuvants for human or animal vaccines are typically subjected to rigorous standards

Span-85

cholesterol



Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in 10:90 acetonitrile:reagent alcoholGradient: Time, %B: -5, 35; 0, 35; 7, 80; 12, 80.Sample Prep.:ABISCO-100™ (ISCONOVA, Uppsala Sweden) was diluted 5-fold with Milli-Q® water and transferred to a glass HPLC autosampler vial.

AddaVax



8.0

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alpha-tocopherol

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100

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60

of analysis including quantification of strength, purity, stability, and degradation behavior, even though they are not currently regulated in the same manner as active pharmaceutical ingredients in the United States. Complicating such analysis, manyadjuvants under investigation contain components that are not readily analyzed bytraditional HPLC with UV detection. These include various mixtures of lipids, fattyacids, and glycosides that lack suitable UV chromophores. In this work, the lack of a detectable chromophore in several adjuvant compounds and degradation products wasovercome by using HPLC with charged aerosol detection, a detector that can measure

cholesterol


lyso‐PC COP



Column: Thermo Scientific Accucore™ C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °C Flow Rate: 0.80 mL/minInjection Vol.: 10 µL Mobile Phase A: 1 mM ammonium acetate in waterMobile Phase B: 2-propanol, Fisher Scientific™ Optima™2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.: 2.0

3.0

4.0

5.0

6.0

7.0 squalene

20

30

40

50

60

70Performance




Adjuvant mixtures and individual components were analyzed by UHPLC employing octylsilyl (C8) and perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or 2.6 micron solid core silica particles. Analytes were detected by bothcharged aerosol detection and diode array detection.






Squalene-tocopherol-PS80 mixtureColumn: Thermo Scientific Accucore PFP 2.6 µm, 2.1× 100 mm Column Temp: 45 °C Flow Rate: 0.50 mL/minInjection Vol : 1 or 3 µL

sMPLA


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suited for the determination of any nonvolatile analyte independent of chemicalcharacteristics. As shown in Figure 1, the detector nebulizes the mobile phase to create aerosol droplets. The mobile phase evaporates in the drying tube, leaving analyte particles, which become charged in the mixing chamber. The charge is then measured by a highly sensitive electrometer, providing reproducible, nanogram-levelsensitivity. This technology has greater sensitivity and precision than evaporative lightscattering detection and refractive index detection and it is simpler to operate than a mass spectrometer.






HPLC eluent

Nebulization and

Drying tube

Signal out

Injection Vol.: 1 or 3 µL Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.08% formic acid in 2-propanol, Optima 2 LC/MSGradient: Time, %B: -5, 30; 0, 30; 13, 95; 18, 95 Sample Prep.:Dissolve 24.3 mg DL-α-tocopherol (Acros Organics, NJ) in 24.3 mL 2-propanol. Dissolve 48.7 mg squalene in 9.64 mL 2-propanol; dilute 5-fold with 2-propanol. Combine 214 µL squalene solution, 237 µL DL-α-tocopherol solution, 97 µL polysorbate 80 and 452 µL of 2 propanol




ug/mL

0 50 100 150 200 250 300Amount

0.00

0.50

1.00

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Synthetic MPLAColumn: Thermo Scientific Accucore C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °C Flow Rate: 0.80 mL/minInjection Vol.: 10 µL Mobile Phase A: 1 mM ammonium acetate in waterM bil Ph B 2 l O ti 2 LC/MS

p y y gdetection.


Ret. Time1

(%RSD)Peak Area1

(%RSD)LOD2

µg/mLR2*

Saponins 78.2 0.06 0.36 13.0 0.9998

Cholesterol 56.8 0.07 0.39 6.4 0.9999

DPPC 60 2 0 07 1 27 4 4 1 000025.0

30.0

35.0

40.0

45.0

50.0pA mAU

25.0

30.0

35.0

40.0

45.0

50.0


AddaVax is a trademark of InvivoGen, San Diego, CA. AbISCO-100 is a registered trademark of ISCONOVA, Uppsala, Sweden. Span 85 and Tween 80 are trademarks of Croda International Plc. Milli-Q is a registered trademark of EMD Millipore Corporation. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.


AddaVax


Liquid waste


Mixing chamber

Ion trap


Mobile Phase B: 2-propanol, Optima 2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.:Synthetic MPLA (Avanti Polar Lipids, GA) was dissolved in chloroform:methanol:water (CMW) 80:20:4 at a concentration of about 1 mg/mL and transferred to a glass HPLC autosampler vial.


2 Hubaux-Vos method


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sMPLA



Time [min]








Charged Aerosol120

140

160

squalene

800

900

1000

ResultsAbISCO-100

AbISCO-100 is a suspension of purified saponins from Quillaja saponaria, cholesterol from sheep wool and egg phosphatidyl choline in phosphate buffered saline1. As seen in Figure 2, all components elute within 12 min from the Hypersil GOLD PFP column






Data Analysis


Saponins + +

Cholesterol + +




40

60

80

100

200

300

400

500

600

700

20.0pA mAU

200

with good resolution. All components and several degradation products including cholesterol oxidation products (COP) and lyso-PC are detected by the charged aerosol detector, whereas some, such as DPPC, show poor response by UV detection.




Introduction

AddaVax

polysorbate 80

Data System 7.1 SR1





Squalene + +

α-Tocopherol + +

sMPLA + +



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AddaVax



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cholesterol


lyso‐PC COP




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7.0 squalene

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70Performance

Calibration curves for the three major components of AbISCO (analyzed in duplicate) are presented in Figure 3. The data were fit to a quadratic equation, yielding coefficients of determination, R2, greater than 0.999 for all three analytes.

Table 1 presents a summary of the method’s performance, including precision of retention time and peak area, the coefficient of determination, and the limits of detection for the three major components of AbISCO.









sMPLA


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j p


3.50

4.00

4.50

5.00


5.00

6.00


4.00

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5.00

5.50

6.00

Figure 3. Calibration data for analysis of AbISCO by HPLC-charged aerosol detection.







HPLC eluent

Nebulization and

Drying tube

Signal out





ug/mL

0 50 100 150 200 250 300Amount

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Area

ug/mL

0 50 100 150 200 250 300 350Amount

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0 50 100 150 200 250 300 350 400 450Amount

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p y y gdetection.


Ret. Time1

(%RSD)Peak Area1

(%RSD)LOD2

µg/mLR2*

Saponins 78.2 0.06 0.36 13.0 0.9998

Cholesterol 56.8 0.07 0.39 6.4 0.9999

DPPC 60 2 0 07 1 27 4 4 1 000025.0

30.0

35.0

40.0

45.0

50.0pA mAU

25.0

30.0

35.0

40.0

45.0

50.0


AddaVax is a trademark of InvivoGen, San Diego, CA. AbISCO-100 is a registered trademark of ISCONOVA,Uppsala, Sweden. Span 85 and Tween 80 are trademarks of Croda International Plc. Milli-Q is a registered trademark of EMD Millipore Corporation. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.


AddaVax


Liquid waste


Mixing chamber

Ion trap




2 Hubaux-Vos method


0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-5.0

0.0

5.0

10.0

15.0

20.0

min-5.0

0.0

5.0

10.0

15.0

20.0


sMPLA


AddaVax, shown in Figure 4, is prepared by emulsification of sorbitan trioleate (SPAN™ 85) in squalene oil (5% v/v) and polysorbate 80 (0.5% w/v) in sodium citrate buffer (10 mM pH 6.5)2.

Time [min]

5Thermo Scientific Poster Note • PN70333_e 11/12S



Table 2. Summary of immunologic adjuvant components investigated in this study. (+) annotation indicates applicable detector.Methods




Charged Aerosol120

140

160

squalene

800

900

1000

ResultsAbISCO-100

AbISCO-100 is a suspension of purified saponins from Quillaja saponaria, cholesterol from sheep wool and egg phosphatidyl choline in phosphate buffered saline1. As seen in Figure 2, all components elute within 12 min from the Hypersil GOLD PFP column


OverviewPurpose: To develop fast and sensitive HPLC methods suitable to measure the purity of immunological adjuvant formulations.

Methods: Adjuvant mixtures and individual components were analyzed by ultra-high performance liquid chromatography (UHPLC) employing octylsilyl (C8) and



Data Analysis

CDS: Thermo Scientific Dionex Chromeleon™ Chromatography Data System 7 1 SR1

Saponins + +

Cholesterol + +




40

60

80

100

200

300

400

500

600

700

20.0pA mAU

200

with good resolution. All components and several degradation products including cholesterol oxidation products (COP) and lyso-PC are detected by the charged aerosol detector, whereas some, such as DPPC, show poor response by UV detection.


perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or 2.6 micron solid core silica particles. Analytes were detected by both charged aerosol detection and photodiode array detection.

Results: Charged aerosol detection is shown to be suitable for measuring components including triterpenoid saponins, cholesterol, phospholipids, and surfactants with sensitivity often superior to that obtained by UV absorbance detection.

Introduction

AddaVax

polysorbate 80

Data System 7.1 SR1


2.1 × 100 mmColumn Temp: 45 °C Flow Rate: 0.47 mL/minInjection Vol.: 10 µL Mobile Phase A: 0 1% formic acid in water



Squalene + +

α-Tocopherol + +

sMPLA + +



0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0Time [min]

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100

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180IntroductionA vaccine adjuvant is any substance that helps promote the effectiveness of a vaccine by reducing the amount or frequency of the required dose, by prolonging the duration of immunological memory, or by modulating the involvement of humoral or cellular responses. This functional definition of adjuvants encompasses a very diverse group of substances whose chemical structures and mechanisms of action vary widely. Adjuvants for human or animal vaccines are typically subjected to rigorous standards

Span-85

cholesterol



Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in 10:90 acetonitrile:reagent alcoholGradient: Time, %B: -5, 35; 0, 35; 7, 80; 12, 80.Sample Prep.:ABISCO-100™ (ISCONOVA, Uppsala Sweden) was diluted 5-fold with Milli-Q® water and transferred to a glass HPLC autosampler vial.

AddaVax



8.0

9.0

10.0

alpha-tocopherol

pA pA

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90

100

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min-10

0

20

40

60

of analysis including quantification of strength, purity, stability, and degradation behavior, even though they are not currently regulated in the same manner as active pharmaceutical ingredients in the United States. Complicating such analysis, many adjuvants under investigation contain components that are not readily analyzed by traditional HPLC with UV detection. These include various mixtures of lipids, fatty acids, and glycosides that lack suitable UV chromophores. In this work, the lack of a detectable chromophore in several adjuvant compounds and degradation products was overcome by using HPLC with charged aerosol detection, a detector that can measure

cholesterol


lyso‐PC COP

Charged aerosol detection enables sensitive measurement of adjuvant components not amenable to detection by UV absorbance. Detection limits for saponins, cholesterol, and DPPC were in the low µg/mL (ng on-column) range.


Column: Thermo Scientific Accucore™ C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °C Flow Rate: 0.80 mL/minInjection Vol.: 10 µL Mobile Phase A: 1 mM ammonium acetate in waterMobile Phase B: 2-propanol, Fisher Scientific™ Optima™2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.: 2.0

3.0

4.0

5.0

6.0

7.0 squalene

20

30

40

50

60

70Performance

Calibration curves for the three major components of AbISCO (analyzed in duplicate) are presented in Figure 3. The data were fit to a quadratic equation, yielding coefficients of determination, R2, greater than 0.999 for all three analytes.

Table 1 presents a summary of the method’s performance, including precision of retention time and peak area, the coefficient of determination, and the limits of detection for the three major components of AbISCO.


Adjuvant mixtures and individual components were analyzed by UHPLC employing octylsilyl (C8) and perfluorophenyl (PFP) stationary phases on either sub-2 micron porous silica or 2.6 micron solid core silica particles. Analytes were detected by both charged aerosol detection and diode array detection.






Squalene-tocopherol-PS80 mixtureColumn: Thermo Scientific Accucore PFP 2.6 µm, 2.1× 100 mm Column Temp: 45 °C Flow Rate: 0.50 mL/minInjection Vol : 1 or 3 µL

sMPLA


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Figure 3. Calibration data for analysis of AbISCO by HPLC-charged aerosol detection.

suited for the determination of any nonvolatile analyte independent of chemical characteristics. As shown in Figure 1, the detector nebulizes the mobile phase to create aerosol droplets. The mobile phase evaporates in the drying tube, leaving analyte particles, which become charged in the mixing chamber. The charge is then measured by a highly sensitive electrometer, providing reproducible, nanogram-level sensitivity. This technology has greater sensitivity and precision than evaporative light scattering detection and refractive index detection and it is simpler to operate than a mass spectrometer.


1. Picard, M.D.; Cohane, K.P.; Gierahn, T.M.; Higgins, D.E.; Flechtner, J.B. High-throughput proteomic screening identifies Chlamydia trachomatis antigens that are capable of eliciting T cell and antibody responses that provide protection against vaginal challenge. Vaccine. 2012, 30(29):4387-93.

2. Mbow M.L.; De Gregorio, E.; Valiante, N.M.; Rappuoli, R. New adjuvants for human vaccines. Curr Opin Immunol. 2010, 2(3):411-6.



HPLC eluent

Nebulization and

Drying tube

Signal out

Injection Vol.: 1 or 3 µL Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.08% formic acid in 2-propanol, Optima 2 LC/MSGradient: Time, %B: -5, 30; 0, 30; 13, 95; 18, 95 Sample Prep.:Dissolve 24.3 mg DL-α-tocopherol (Acros Organics, NJ) in 24.3 mL 2-propanol. Dissolve 48.7 mg squalene in 9.64 mL 2-propanol; dilute 5-fold with 2-propanol. Combine 214 µL squalene solution, 237 µL DL-α-tocopherol solution, 97 µL polysorbate 80 and 452 µL of 2 propanol


y p p y p , g p p y ( ),low toxicity while specifically activating TLR4 but not TLR2. sMPLA contains six acyl groups. Purity analysis is used to quantify contaminants, degradation products, and variants differing in acyl chain number, length, and phosphorylation3. Figure 6 compares chromatograms obtained from analysis of sMPLA by inverse gradient HPLC with detection by UV absorbance at 220 nm or charged aerosol detection.


ug/mL

0 50 100 150 200 250 300Amount

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Synthetic MPLAColumn: Thermo Scientific Accucore C8 2.6 µm, 4.6 × 150 mm Column Temp: 40 °C Flow Rate: 0.80 mL/minInjection Vol.: 10 µL Mobile Phase A: 1 mM ammonium acetate in waterM bil Ph B 2 l O ti 2 LC/MS

p y y gdetection.


Ret. Time1

(%RSD)Peak Area1

(%RSD)LOD2

µg/mLR2*

Saponins 78.2 0.06 0.36 13.0 0.9998

Cholesterol 56.8 0.07 0.39 6.4 0.9999

DPPC 60 2 0 07 1 27 4 4 1 000025.0

30.0

35.0

40.0

45.0

50.0pA mAU

25.0

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45.0

50.0

sMPLA We would like to thank Isconova, Uppsala, Sweden for making available the AbISCO immunological reagent.



AddaVax


Liquid waste


Mixing chamber

Ion trap


Mobile Phase B: 2-propanol, Optima 2 LC/MSGradient: Time, %B: 0, 65; 3, 65; 13, 90; 18, 90; 18.1, 65; 30, 65.Sample Prep.:Synthetic MPLA (Avanti Polar Lipids, GA) was dissolved in chloroform:methanol:water (CMW) 80:20:4 at a concentration of about 1 mg/mL and transferred to a glass HPLC autosampler vial.


2 Hubaux-Vos method


0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-5.0

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AddaVax, shown in Figure 4, is prepared by emulsification of sorbitan trioleate (SPAN™ 85) in squalene oil (5% v/v) and polysorbate 80 (0.5% w/v) in sodium citrate buffer (10 mM pH 6.5)2.

Time [min]




Table 2. Summary of immunologic adjuvant components investigated in this study. (+) annotation indicates applicable detector.Methods




Charged Aerosol120

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squalene

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1000

ResultsAbISCO-100







Data Analysis


Saponins + +

Cholesterol + +




40

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Introduction

AddaVax

polysorbate 80

Data System 7.1 SR1





Squalene + +

α-Tocopherol + +

sMPLA + +



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Span-85

cholesterol


precision better than 0.1% RSD and peak area precision between 0.4 and 1.3%RSD for the major components.


AddaVax



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pA pA

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cholesterol


lyso‐PC COP




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sMPLA


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1. Picard, M.D.; Cohane, K.P.; Gierahn, T.M.; Higgins, D.E.; Flechtner, J.B. High-throughput proteomic screening identifies Chlamydia trachomatis antigens that are capable of eliciting T cell and antibody responses that provide protection against vaginal challenge. Vaccine. 2012, 30(29):4387-93.




HPLC eluent

Nebulization and

Drying tube

Signal out





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p y y gdetection.


Ret. Time1

(%RSD)Peak Area1

(%RSD)LOD2

µg/mLR2*

Saponins 78.2 0.06 0.36 13.0 0.9998

Cholesterol 56.8 0.07 0.39 6.4 0.9999

DPPC 60 2 0 07 1 27 4 4 1 000025.0

30.0

35.0

40.0

45.0

50.0pA mAU

25.0

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sMPLA We would like to thank Isconova, Uppsala, Sweden for making available the AbISCO immunological reagent.



AddaVax


Liquid waste


Mixing chamber

Ion trap




2 Hubaux-Vos method


0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-5.0

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sMPLA



Time [min]

www.thermofisher.com/dionex©2016 Thermo Fisher Scientific Inc. All rights reserved. AddaVax is a trademark of InvivoGen, San Diego, CA. AbISCO-100 is a registered trademark of ISCONOVA, Uppsala, Sweden. Span 85 and Tween 80 are trademarks of Croda International Plc. Milli-Q is a registered trademark of EMD Millipore Corporation. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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direct analysis of multicomponent adjuvants by hplc with ......of analysis including quantification...

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